Biology Lab Practical Midterm Study Guide PDF

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Biology Lab study guide notes for the practical/midterm....

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FOCUS ON: - Analyzing/interpreting data in Excel - Determining concentration of unknowns - Calculating enzyme rate - Technical skills - Pipetting - Spectrovis - Dilutions - WQI tests - Using lab equipment Serial Dilutions - C1V1 = C2V2 - V2 (total) - V1 = V (water or buffer) - Cs (concentration of standard) / Cu (concentration of unknown) = As (absorption of standard) / Au (absorption of unknown) - C1/C2 = SDF - Total V / SDF = transfer volume Serial Dilutions Pt. 2 - Measuring Absorbance - Go to LabQuest app, click on Sensors, go to Calibrate - After the 90 second warm up, put in the blank cuvette, press finish calibration - MAKE SURE to go to Sensors, Data Collection, and change the mode to TIME  BASED - Tap on the big red box on the screen and choose Select Wavelength and change the absorbance to whatever is given or 550 nm, press OK - Put in cuvette 0 in place of the blank and press the play button - Wait at least 10 seconds and press the stop button - Go to Analyze in the top of the screen and click Statistics and record the average (mean) absorbance. - Put in cuvette 1 in place of cuvette 0 and press the play button again and discard the data when asked - Repeat Enzyme rate = absorbance / time Reaction rate = (absorbance final - absorbance initial) / (time final - time initial)

Calculating pH - Plug the pH probe into channel 1 of LabQuest - Rinse the pH probe with distilled water and gently wipe with a Kimwipe - Submerge the pH probe into whatever you’re testing - Wait 15 seconds before collecting data to let the pH probe get used to the new environment - Press the play button on LabQuest and collect data for 10 seconds - Stop data by pressing the stop button - Go to Analyze in the top of the screen and select Statistics and record the average pH Calculating Temperature - Plug the temperature probe into channel 1 of LabQuest - Bring the probe and the test buffer around the room and stick it into each different “environment” (ice, benchtop, warm water, hot water) - Press the play button and collect data for 10 seconds - Stop data collection by pressing the stop button - Go to Analyze at the top of the screen and select Statistics and record the average temperature Total Solids - If the mass of the solids is at least 0.025 g after subtracting final - initial mass, proceed Biochemical Oxygen Demand (BOD) - Collect 3 water samples with 3 different BOD bottles - Plug the dissolved oxygen probe into channel 1 of LabQuest - Rinse the tip of the probe with distilled water and gently wipe down with a Kimwipe - Set the switch on the DO probe to collect data in mg/L  - Submerge the BOD bottle and hold it there for 40 seconds - Begin data collection by pressing the play button and collect data for 10 seconds - Stop data collection/press the stop button - Determine the average DO concentration by going to Analyze at the top of the screen, then Statistics. Record this data under “initial dissolved oxygen” - While still submerged, flip the switch on the DO probe to % and measure (press play) for 10 seconds. Record the average % saturation Dissolved Oxygen (DO) - Use the initial DO readings from your BOD bottles for the DO value

Nitrates - Attach the probe clamp to the support stand - Carefully insert the Nitrate ISE to the probe clamp and make sure that it is set up vertically - Uncap the Nitrate High Standard and place it on the support stand, directly under the Nitrate ISE - Lower the probe clamp so that the Nitrate ISE is submerged in the Nitrate High Standard but NOT touching the bottom of the bottle - Soak the Nitrate ISE in the Nitrate High Standard solution for 30 min. - Plug the ISE sensor into channel 1 of the vernier interface - Go to Sensors in the menu bar at the top of the screen, select Calibrate, select Nitrate ISE - Select Calibrate Now - Wait for the “live voltage” reading to stabilize. Then enter 100 for Value 1 and press Keep - Swap out the Nitrate High Standard for the rinsate beaker - Rinse thoroughly - Swap out the rinsate beaker for the Nitrate Low Standard - Lower the probe clamp so that the Nitrate ISE is submerged into the Low Standard - Wait for the “live voltage” reading to stabilize. Then enter 1 for Known Value 2 and press Keep - Press OK to finish the calibration - Rinse the ISE again - Place the tip of the ISE into the sample water in the Nalgene bottle - Leave the probe submerged for 60 seconds before collecting data - Press play, collect data for 10 seconds - Stop data collection, go to Analyze, then Statistics, and record the average nitrate concentration Total Phosphate - Calibrate the SpectroVis to use for measuring the absorbances - Connect the SpectroVis to the LabQuest via the USB cable - Go to Sensors at the top of the screen, select Calibrate and select Spectrometer. Wait for the 90 second warm up and place the blank in the cuvette holder of the SpectroVis, and press Finish Calibration. Press OK - Go to Sensors and select Data Collection and change the mode from “Full Spectrum” to “Time Based”, press OK - Tap on the big red box and select Change Wavelength from the drop down menu. Enter the given absorbance or 565 nm and press OK.

WRITTEN PORTION Proteins - its activity is often very dependent on pH, temperature, and salt concentration of the reaction mixture. Buffers - an aqueous solution mixture, function to resist changes in hydrogen ion concentration. Stock Solutions - concentrated solutions that last over a long period of time Dilution Factor - factor by which the concentration of the dilute solution is reduced compared to the concentration of the stock solution. Serial Dilution - stepwise dilution where the stock solution for each dilution is the dilute solution from the previous dilution. Spectrophotometer - a machine that can measure absorbance or transmittance of a pigmented solution. Standards (solution) - have a known amount of the material being assayed and are used to calculate the amount of material in the unknowns. Standard Curve - tool used to determine concentration of unknown. Independent Variable - on the X-axis. Dependent Variable - on the Y-axis. Discovery Science - uses large amounts of data or surveys of natural systems to discover patterns and correlations. Hypothesis-driven Science - uses the scientific method to develop knowledge. Scientific Method 1. The problem or observation 2. Collect background information 3. State the hypothesis 4. State predictions 5. Test predictions (with experiment) 6. Draw a conclusion

7. Report the conclusion Cytochrome C Oxidase - converts reduced cytochrome c to oxidized cytochrome c, complex IV of the electron transport chain. The electrons pass through this complex and bond with two protons (H+) in the surrounding aqueous solution to form water. This enzyme is highly conserved in organisms because it is required for mitochondrial respiration. The rate of this reaction is the amount of color change over a specified time period. Reduced cytochrome C oxidase is pink, oxidized cytochrome C oxidase is dark red. DELTA ABSORBANCE / DELTA TIME = RATE OF REACTION Descriptive Statistics - used to describe and summarize data. Mean, median, mode are measures of central tendency and range, variance, standard deviation are measures of dispersion. Inferential Statistics - used to make estimates and draw conclusions, or inferences, about the population based on the sample. T-Test is one example. Temperature Effects on WQI - Cold water = more dissolved gas or DO - Over one mile of a stream, if the temperature changes by even a few degrees, it could indicate a source of thermal pollution. - Increased temperature of water can increase photosynthesis in aquatic plants and algae, leading to increased plant growth and algal blooms. Algal blooms are BAD for the ecosystem. - Thermal pollution - caused by human activities like using river water, and treating it before returning it to the river. The temperature is now warmer than before. - Runoff - from parking lots and rooftops, is warmer than the stream and will increase the overall temperature. - Shade - very important to the health of a stream because it blocks too much direct sunlight. - Removing shade from trees - caused by humans. Increases the water temperature - More shallow streams are more susceptible to changes in temperature - Water can be cooled by cooler air temperatures or colder water from a spring or a tributary. - Organisms become stressed in extreme high or low temperatures, lowering their resistance to pollutants, parasites, and diseases. - Cooler water is BETTER THAN warmer water for aquatic organisms - Most organisms have an optimal temperature range below 25 degrees Celsius, above 0 degrees.

pH Effects on WQI - Small changes can endanger many types of animals - Algal Blooms - very basic, changes pH - Industrial processes - may release acids or bases, changing pH - Oxidation of sulfide-containing sediments - very acidic, changes pH - Rainfall = slightly acidic, but react with minerals in the soil and increase pH of streams and lakes. - pH value of streams and lakes are usually between 7 and 8 - Most drinking water has pH between 6.5 and 8.5 - 6.5-8.2 optimal for most organisms Total Solids Effects on WQI - Soil erosion - increase particles like clay and silt, or dissolved rocks and minerals - Runoff - includes fertilizers and soil particles or industrial wastes. - Plankton, plant and animal matter, catfish (bottom-dwelling organism) - add to total solids - Dissolved Solids - add to total solids, sometimes more dissolved solids are present than suspended particles. - HIGH total solids - makes the water cloudy so less photosynthesis. More cloudiness also increases temperature, resulting in more problems. - Too many dissolved salts in water can dehydrate organisms. Too little can limit the growth of aquatic organisms. - Usually fall within the range of 20 mg/L to 500 mg/L. Dissolved Oxygen Effects on WQI - Higher levels of DO = more variety of organisms in the water - Oxygen is dissolved by diffusion bw the atmosphere and water at its surface, aeration as water flows over rocks and other debris, churning of water by waves and wind, photosynthesis of aquatic plants. - FACTORS THAT AFFECT DO CONCENTRATIONS: - Temperature - Stream flow - Air pressure - Aquatic plants - Decaying organic matter - Human activities

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Algal Bloom - increases dissolved oxygen levels but dies, and when the algae die, aerobic decomposers use up all the oxygen. This causes a sharp drop in DO and organisms can suffocate and die right after an algal bloom. Concentrations can range from 0 to 15 mg/L Often measured by PERCENT SATURATION

Biochemical Oxygen Demand (BOD) Effects on WQI - In a healthy stream, oxygen is replenished faster than it is used by organisms - Has to do with rate of aerobic decomposers using oxygen and rate of oxygen being produced by plants - Too much organic material to decompose = high BOD. - Initial DO - final DO = BOD level - Lower BOD level = healthier water Nitrates Effects on WQI - Necessary for plants and animals to synthesize amino acids and proteins - Can be increased by fertilizers, legume-plant nitrogen fixation, plant and animal decomposition, animal waste, runoff from fertilized fields, treated wastewater being returned to streams, animal feedlots - Healthy (unpolluted) water has less than 1 mg/L - Too many nitrates (above 10 mg/L) in drinking water can cause Blue Baby Syndrome in infants (methemoglobinemia) - High levels can also cause eutrophication. Total Phosphate Effects on WQI - Only a small amount needed as the limiting factor in plant and algal growth. - Too much = eutrophication - High levels of phosphates are associated with decreased DO and increased BOD - Four classifications of phosphates: - Orthophosphates: inorganic forms of phosphates used heavily in fertilizers and often introduced through runoff - Organically bound phosphates: found in human and animal wastes or in decaying matter - Condensed phosphates (polyphosphates): sometimes added to water supplies and industrial processes - Total phosphates: sum of all three forms above. Most commonly reported form of phosphate concentration - Phosphates are added to water through humans (industrial/agricultural waste), fertilizers through runoff and soil erosion, excrement of animals living in or near the water.

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Levels above 0.1 mg/L can stimulate plant growth above its natural rate (BAD)

Fecal Coliform Bacteria, WQI - Indicates other pathogens from feces such as disease-causing bacteria, viruses and parasites - Could be caused by leaking septic or sewer systems, polluted runoff that has picked up animal waste on the way to the stream, waterfowl, wading cows. - Coliform bacteria are facultative anaerobes that can tolerate oxygen but can survive without it. Ferments lactose to produce gas. - Durham tubes testing for fecal coliform is incubated at 44.5 degrees celsius (higher than other coliforms’ optimal temperature) so the other coliforms other than fecal coliforms will be unable to survive. Turbidity, WQI - Factors that increase turbidity: increase in stream flow from heavy rains, soil erosion, runoff (agricultural, industrial wastes, water treatment plant effluent, urban runoff from parking lots/roads/rooftops) - Bottom dwelling organisms, decaying matter, plankton can cause turbidity - Very similar effects to WQI as Total Solids (less clear water = less photosynthesis, more solids = warmer temp. of water) - Standard for drinking water is 0.5 NTU to 1.0 NTU - Turbidity is obviously visible at levels above 5 NTU Conserved Region of DNA - a nucleotide sequence that has little to no variation across species and has remained constant throughout evolution DNA Barcoding - Purify the genomic DNA sample to prepare for amplification during PCR - For Bacteria: - Agar serves as a growth medium for bacteria culture - DO NOT NEED a lysis buffer for bacterial samples because bacteria is prokaryotic (no membrane bound organelles) so the heat used in PCR will lyse the cells on its own. - For Animals/Plants: - Break down the tissue in a bead tube homogenizer → chemical/enzymatic digestion → add lysis solution to help lyse the cells - Lysis solution : contains EDTA, helps break down membranes by denaturing them and helps to inactivate nucleases

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Animal lysis solution: also contains RNaseA, which breaks down RNA

DNA Purification/Isolation - Denature: heat the sample to 94 degrees Celsius for 5 min. - Purify the DNA (bacterial DNA can be used directly, without this step) - Centrifuge lysate already made - DNA will bind to column because of the ionic interaction b/w DNA and the salt on the column - Wash buffer contains ethanol, clears everything away but DNA is not soluble in ethanol so it will stay stuck to the column - Then nuclease free water is added to the column and centrifuged again. DNA is water soluble so it will be pulled through the column and be isolated. (FINAL WASH, THERE ARE 4 WASHES) Polymerase Chain Reaction (PCR) 1. Denaturation: the purified genomic DNA is placed in a microcentrifuge for 1 min at 95 degrees celsius. Disrupts the hydrogen bonds holding the strands together and makes single stranded DNA. 2. Annealing: Two primers (For. and Rev.) anneal at 5’ end and 3’ end at 50 degrees celsius for one min. 3. Synthesis: Temperature is raised to 72 degrees celsius to allow Taq polymerase to synthesize DNA from the two primers. - EACH PCR CYCLE GENERATES TWO COPIES (DOUBLES) 2^n - The two primers used are called Forward and Reverse. Master Mix - Consists of DNA polymerase, dNTPs, buffer, forward & reverse primers, water needed to perform multiple PCR reactions. - Helps reduce pipetting errors and time spent running the PCR reaction - Pipette the master mix into a PCR tube before adding an aliquot (small portion) of genomic DNA or bacterial culture to the PCR tube. Gel Electrophoresis - Standard method used to separate, identify, and purify DNA fragments - DNA molecules migrate toward the positively charged electrode (red) because of its net negative charge - Smaller fragments of DNA move faster through the porous matrix of agarose - Agarose and a buffer are used to make a melted substance that cools and hardens in a casting tray, forming a jello-like consistency.

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Blue gel loading dye is added to the DNA samples, is consisted of xylene cyanol and bromophenol blue: allow for monitoring of how far the DNA has migrated in the gel. Ethidium bromide (EtBr) is used in the gel to help visualize the DNA fragments, but could possibly distort the DNA upon binding and cause mutations = weak carcinogen. It binds to DNA and shines bright red when exposed to UV light. Run the gel at a voltage of 150 volts Turn off when the purple dye migrated 5 cm COI = 700 bp rbcL = 600 bp 16S = 1500 bp

Gel Analysis - Presence of the ladder indicates: 1) The gel was made properly 2) Electrophoresed in the correct direction 3) Stained by ethidium bromide *NO BANDS (INCLUDING THE LADDER) PRESENT IN THE GEL MEANS ETHIDIUM BROMIDE WAS NOT ADDED* - If the ladder is visible but the sample bands are not there, that means that part of the Taq polymerase may have been degraded - Both Taq polymerase and dNTPs are susceptible to degradation with improper handling and/or storage - No sample band present for any sample type means that one or both of the appropriate primers were not added to the master mix - If there are no bands in animal or plant  lane: 1) It is possible that your DNA extraction recovered little or no genomic DNA 2) It is possible that you did not add 2 microliters of genomic DNA to the PCR - If there is no band in your bacteria  lane: 1) It is possible that you did not properly inoculate your bacteria culture 2) It is possible that you did not add 2 microliters of bacterial culture to the PCR - Multiple PCR bands: 1) If there is a second location in the template where primers can anneal, a second band will show up 2) There are several causes: - Non-specific primer binding: primers randomly hybridize to the genomic DNA at different locations and initiate synthesis. Can be caused by sub-optimal primer design or contamination of either the DNA or PCR samples with DNA from another class of organisms (ex: plant DNA contaminated by animal DNA)

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For COI primer set, if the animal genomic DNA or animal COI  PCR sample is contaminated with either plant or bacterial DNA, you will NOT see a second band on the gel-- the COI  primer set always produces a 700 bp PCR products for all plants and some bacteria if there is enough template COI primer set could have been added to bacterial or plant master mix

Finch TV - Good sequences have wavelengths that are roughly the same height, symmetrical, and non-overlapping - Adenine, Thymine, Guanine, Cytosine - Quality Value - grey bars at the top of each wavelength -- accesses the accuracy of each base in a DNA sequence - BLASTN - used to search the nucleotide database using a nucleotide query - Nucleotide query = cropped, edited sequence - BLASTP - compares an amino acid (protein) query sequence against a protein sequence database - BLASTX - compares a nucleotide query sequence translated in all reading frames against a protein sequence database - TBLASTN - compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames - TBLASTX - compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database - First crop thefd end of the sequence (3’ end) and then the beginning of the sequence (5’ end) - Heterozygous peaks - two different colored wavelengths overlapped, caused by “noise” - Max score - based on the extent and quality of the match - Query cover - shows how much of the query sequence is matched to the sequence of the database - E Value - a parameter that describes the number of hits one can "expect" to see this good by chance when searching a database of a particular size. The closer the value is the 0, the greater the likelihood that the match is meaningful/not due to chance - Sequences with high identi...